chromatin modifications and their function
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Leading Edge
Review
Chromatin Modifications and Their FunctionTony Kouzarides1,*1The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB21QN, UK
*Correspondence: t.kouzarides@gurdon.cam.ac.uk
DOI 10.1016/j.cell.2007.02.005
The surface of nucleosomes is studded with a multiplicity of modifications. At least eightdifferent classes have been characterized to date and many different sites have been iden-tified for each class. Operationally, modifications function either by disrupting chromatincontacts or by affecting the recruitment of nonhistone proteins to chromatin. Their presenceon histones can dictate the higher-order chromatin structure in which DNA is packaged andcan orchestrate the ordered recruitment of enzyme complexes to manipulate DNA. In thisway, histone modifications have the potential to influence many fundamental biologicalprocesses, some of which may be epigenetically inherited.
Chromatin is the state in which DNA is packaged within
the cell. The nucleosome is the fundamental unit of chro-
matin and it is composed of an octamer of the four core
histones (H3, H4, H2A, H2B) around which 147 base pairs
of DNA are wrapped. The core histones are predominantly
globular except for their N-terminal ‘‘tails,’’ which are
unstructured. A striking feature of histones, and particu-
larly of their tails, is the large number and type of modified
residues they possess. There are at least eight distinct
types of modifications found on histones (Table 1). We
have the most information regarding the small covalent
modifications acetylation, methylation, and phosphoryla-
tion. However this Review tries to encompass as thor-
oughly as possible all modifications of the core histones,
concentrating on recent literature. It covers the enzymes
that mediate modifications, their mechanism of action,
and their biological function. In the first few sections,
some general issues regarding the analysis modifications
are discussed along with some general principles regard-
ing their mechanism of action. Each class of modification
is then reviewed more specifically under the heading of
the function it regulates. The ‘‘Functions Regulated’’
part of Table 1 should act as a guide as to where a modifi-
cation is mentioned in detail. At the end of this Review, the
epigenetic nature of modifications is discussed.
Characterizing Histone Modification
Histones are modified at many sites. There are over 60 dif-
ferent residues on histones where modifications have
been detected either by specific antibodies or by mass
spectrometry. However, this represents a huge underesti-
mate of the number of modifications that can take place
on histones. Extra complexity comes partly from the fact
that methylation at lysines or arginines may be one of three
different forms: mono-, di-, or trimethyl for lysines and
mono- or di- (asymmetric or symmetric) for arginines.
This vast array of modifications gives enormous potential
for functional responses, but it has to be remembered
that not all these modifications will be on the same histone
at the same time. The timing of the appearance of a
modification will depend on the signaling conditions within
the cell.
The use of modification-specific antibodies in chroma-
tin immunoprecipitations coupled to gene array technol-
ogy (ChIP on CHIP) has revolutionized our ability to mon-
itor the global incidence of histone modifications. Such
global analysis has only been done on a subset of modifi-
cations (acetylation and lysine methylation), but the results
clearly show that modifications are not uniformly distrib-
uted. Most of the information we have has come from
global analyses in budding yeast (Liu et al., 2005; Pokho-
lok et al., 2005). Certain common features have come to
light regarding the composition and enrichment of modifi-
cations on actively transcribed genes: acetylation is
enriched at specific sites in the promoter and 50 end of
the coding regions; within the promoter there are two
nucleosomes flanking the initiation site that are hypo-
acetylated at certain lysines and are enriched in the H2A
variant Hzt1 (Liu et al., 2005; Zhang et al., 2005; Raisner
et al., 2005; Millar et al., 2006; Millar and Grunstein,
2006); the initiation site itself is devoid of nucleosomes;
lysine trimethylation is enriched in the coding region; and
each of the three known methylation sites in yeast
(H3K4, H3K36, H3K79) has a specific distribution pattern.
Thus there is a basic blueprint of modification patterning in
yeast. Limited evidence from mouse and human tissues
indicates that this is a conserved characteristic (Bernstein
et al., 2005; see Review by B.E. Bernstein et al., page 669
of this issue).
However, the ChIP on CHIP approach does have
a shortfall. It can detect the modification status over
a range (2–3) of nucleosomes or even on a single nucleo-
some, but it cannot determine the modification status of
different histones within the same nucleosome. So it is
not possible to determine if both copies of a histone are
identically modified within a single nucleosome or whether
there is a distinct pattern on each. The only way to address
this issue is to use mass spectrometry, but the fact that
Cell 128, 693–705, February 23, 2007 ª2007 Elsevier Inc. 693
Table 1. Different Classes of Modifications Identified on Histones
Chromatin Modifications Residues Modified Functions Regulated
Acetylation K-ac Transcription, Repair, Replication, Condensation
Methylation (lysines) K-me1 K-me2 K-me3 Transcription, Repair
Methylation (arginines) R-me1 R-me2a R-me2s Transcription
Phosphorylation S-ph T-ph Transcription, Repair, Condensation
Ubiquitylation K-ub Transcription, Repair
Sumoylation K-su Transcription
ADP ribosylation E-ar Transcription
Deimination R > Cit Transcription
Proline Isomerization P-cis > P-trans Transcription
Overview of different classes of modification identified on histones. The functions that have been associated with each modification
are shown. Each modification is discussed in detail in the text under the heading of the function it regulates.
a protein has to be digested before such analysis can take
place limits its potential. New methodology that uses
a top-down proteomics approach (identify protein first
and digest subsequently) gives promise that we may, in
the future, look at the intact modification pattern of differ-
ent histones in a given nucleosome (Macek et al., 2006).
Once global analysis of all histone modifications is
done, a prediction would be that every single nucleosome
would be found to be modified in some way. This picture is
of course very static. The truth is that modifications on his-
tones are dynamic and rapidly changing. Acetylation,
methylation, phosphorylation, and deimination can
appear and disappear on chromatin within minutes of
stimulus arriving at the cell surface. Thus examining bulk
histones under one specific set of conditions (with either
antibodies or mass spectrometry) will identify only a
proportion of the possible modifications.
There are also problems of detection that are specific
for antibodies. Firstly, there are the obvious issues of
specificity. These are difficult to avoid as there are no
true controls for modifications in mammalian cells (unlike
yeast) where it is impossible to mutate the residue to
make sure reactivity is lost. In addition, an adjacent
modification may disrupt the binding of the antibody or
a protein may occlude its recognition, both of which may
give a false reading. Similarly, there are problems of
detection that are specific to mass spectrometry. Peptide
coverage is not equivalent for all parts of the histone and
this reduces the sensitivity of detection in these regions.
These facts undoubtedly contribute to our underestima-
tion of the extent of modifications present on histones.
We assume that each individual modification on his-
tones leads to a biological consequence. However proof
of a consequence is not always easy to provide and is
often based on a correlation: a modification appears on
a gene under certain conditions (e.g., when it is tran-
scribed) and disappears when that state is reversed
(e.g., when the gene is silent). Proving causality for a
modification involves showing that the catalytic activity
of the enzyme that mediates the modification is necessary
694 Cell 128, 693–705, February 23, 2007 ª2007 Elsevier Inc
for the biological response. However we know that many
of the histone-modifying enzymes have other nonhistone
substrates. So the response may be going through
another unidentified protein substrate. Furthermore, there
may be signaling redundancy such that more than one
enzyme may be capable of modifying a specific site. In
this case, the effects of inactivating one enzyme may be
masked by an upregulation in the activity of a second
distinct but related enzyme. Showing that mutation of
the modified residue gives the same output as mutating
the enzyme is a second stringent test. However, this is
not possible in humans due to many histone genes
present in the genome, but it is possible in yeast.
So the truth is that we have ‘‘levels of confidence’’
regarding the causative nature of different modifications
depending on how far the analysis has gone to prove the
issue. We also have to be realistic and accept that, how-
ever far we go in proving that a histone modification is
causative, we can never exclude the possibility that
modification of other substrates by the same enzyme
will play a parallel role in the biological response being
monitored. The many other nonhistone substrates of
chromatin-modifying enzymes are not covered in this
Review.
Histone-Modifying Enzymes
The identification of the enzymes that direct modification
has been the focus of intense activity over the last 10 years
(Table 2). Enzymes have been identified for acetylation
(Sterner and Berger, 2000), methylation (Zhang and Rein-
berg, 2006), phosphorylation (Nowak and Corces, 2004),
ubiquitination (Shilatifard, 2006), sumoylation (Nathan
et al., 2006), ADP-ribosylation (Hassa et al., 2006), deimi-
nation (Cuthbert et al., 2004; Wang et al., 2004b), and pro-
line isomerization (Nelson et al., 2006).
Most modifications have been found to be dynamic,
and enzymes that remove the modification have been
identified. One major exception is methylation of
arginines: although they are thought to be dynamic, a
demethylating activity has not yet been found. Instead
.
Table 2. Histone-Modifying Enzymes
Enzymes that
Modify Histones Residues Modified
Acetyltransferase
HAT1 H4 (K5, K12)
CBP/P300 H3 (K14, K18) H4 (K5, K8)
H2A (K5) H2B (K12, K15)
PCAF/GCN5 H3 (K9, K14, K18)
TIP60 H4 (K5, K8, K12, K16)
H3 K14
HB01 (ScESA1, SpMST1) H4 (K5, K8, K12)
ScSAS3 H3 (K14, K23)
ScSAS2 (SpMST2) H4 K16
ScRTT109 H3 K56
Deacetylases
SirT2 (ScSir2) H4 K16
Lysine
Methyltransferase
SUV39H1 H3K9
SUV39H2 H3K9
G9a H3K9
ESET/SETDB1 H3K9
EuHMTase/GLP H3K9
CLL8 H3K9
SpClr4 H3K9
MLL1 H3K4
MLL2 H3K4
MLL3 H3K4
MLL4 H3K4
MLL5 H3K4
SET1A H3K4
SET1B H3K4
ASH1 H3K4
Sc/Sp SET1 H3K4
SET2 (Sc/Sp SET2) H3K36
NSD1 H3K36
SYMD2 H3K36
DOT1 H3K79
Sc/Sp DOT1 H3K79
Pr-SET 7/8 H4K20
SUV4 20H1 H4K20
SUV420H2 H4K20
SpSet 9 H4K20
EZH2 H3K27
RIZ1 H3K9
the process of deimination has been demonstrated to
correlate with the disappearance of methyl-arginines,
indicating that deimination has the potential to antagonize
arginine methylation. There is no known enzyme that will
convent peptidyl citrulline back to arginine, but evidence
exists that this may be possible given the transient
appearance of citrulline on promoters. Proline isomeriza-
tion is by definition reversible as most isomerases have
intrinsic ability to catalyze the formation of both cis-
and trans-proline.
Of all the enzymes that modify histones, the methyl-
transferases and kinases are the most specific. This is per-
haps the reason why methylation is the most character-
ized modification to date. Phosphorylation of histones is
perhaps not as analyzed as methylation because distinct
Table 2. Continued
Enzymes that
Modify Histones Residues Modified
Lysine Demethylases
LSD1/BHC110 H3K4
JHDM1a H3K36
JHDM1b H3K36
JHDM2a H3K9
JHDM2b H3K9
JMJD2A/JHDM3A H3K9, H3K36
JMJD2B H3K9
JMJD2C/GASC1 H3K9, H3K36
JMJD2D H3K9
Arginine Methlytransferases
CARM1 H3 (R2, R17, R26)
PRMT4 H4R3
PRMT5 H3R8, H4R3
Serine/Thrionine Kinases
Haspin H3T3
MSK1 H3S28
MSK2 H3S28
CKII H4S1
Mst1 H2BS14
Ubiquitilases
Bmi/Ring1A H2AK119
RNF20/RNF40 H2BK120
Proline Isomerases
ScFPR4 H3P30, H3P38
Only enzymes with specificity for one or a few sites have been
included, along with the sites they modify. Human and yeastenzymes are shown. The yeast enzymes are distinguished by
a prefix: Sc (Saccharomyces cerevisiae) or Sp (Saccharomyces
pombe). Enzymes that fall within the same family are grouped.
Cell 128, 693–705, February 23, 2007 ª2007 Elsevier Inc. 695
Figure 1. Recruitment of Proteins to Histones
(A) Domains used for the recognition of methylated lysines, acetylated lysines, or phosphorylated serines. (B) Proteins found that associate prefer-
entially with modified versions of histone H3 and histone H4.
signaling pathways need to be activated to observe the
modifications. In some cases, the specificity of enzymes
that modify histones can be influenced by other factors:
complexes in which enzymes are found may specify
a preference for nucleosomal verses free histones (Lee
et al., 2005a); proteins that associate with the enzyme
may affect its selection of residue to modify (Metzger
et al., 2005) or the degree of methylation (mono-, di-, or
tri-) at a specific site (Steward et al., 2006).
Mechanisms of Histone Modification Function
There are two characterized mechanisms for the function
of modifications. The first is the disruption of contacts
between nucleosomes in order to ‘‘unravel’’ chromatin
and the second is the recruitment of nonhistone proteins.
The second function is the most characterized to date.
Thus, depending on the composition of modifications
on a given histone, a set of proteins are encouraged to
bind or are occluded from chromatin. These proteins
carry with them enzymatic activities (e.g., remodeling
ATPases) that further modify chromatin. The need to
recruit an ordered series of enzymatic activities comes
from the fact that the processes regulated by modifica-
tions (transcription, replication, repair) have several steps.
Each one of these steps may require a distinct type of
chromatin-remodeling activity and a different set of
modifications to recruit them. Below is a more detailed
description of the different mechanisms by which modifi-
cations work.
Modifications may affect higher-order chromatin struc-
ture by affecting the contact between different histones in
adjacent nucleosomes or the interaction of histones with
DNA. Of all the known modifications, acetylation has the
most potential to unfold chromatin since it neutralizes
the basic charge of the lysine. This function is not easy
to observe in vivo, but biophysical analysis indicates that
intern-nucleosomal contacts are important for stabiliza-
tion of higher-order chromatin structure. Thus, any alter-
ation in histone charge will undoubtedly have structural
consequences for the chromatin architecture. Further-
more, the recent development of strategies to make
recombinant nucleosomes modified at specific sites has
allowed this question to be addressed in vitro. By chemi-
cally ligating modified tail peptides onto recombinant
histone core preparations, it has been possible to show
696 Cell 128, 693–705, February 23, 2007 ª2007 Elsevier Inc.
that acetylation of H4K16 has a negative effect on the
formation of a 30-nanometer fiber and the generation
of higher-order structures (Shogren-Knaak et al., 2006;
also see Minireview by D. Trementhick, page 651 of this
issue). Phosphorylation is another modification that may
well have important consequences for chromatin com-
paction via charge changes. The role of this modification
has not been demonstrated rigorously in vitro but demon-
strations of it’s role in mitosis, apoptosis, and gametogen-
esis are suggestive of such a role (Ahn et al., 2005; Fischle
et al., 2005; Krishnamoorthy et al., 2006).
Proteins are recruited to modifications and bind via
specific domains (Figure 1A). Methylation is recognized
by chromo-like domains of the Royal family (chromo,
tudor, MBT) and nonrelated PHD domains, acetylation is
recognized by bromodomains, and phosphorylation is
recognized by a domain within 14-3-3 proteins.
A number of proteins have been identified that are re-
cruited to specific modifications (Figure 1B). The recent
isolation of several proteins that recognize H3K4me has
highlighted the fact that their purpose is to tether enzy-
matic activities onto chromatin. BPTF, a component of
the NURF chromatin-remodeling complex, recognizes
H3K4me3 via a PHD domain. This recruitment tethers
the SNF2L ATPase to activate H0XC8 gene expression
(Wysocka et al., 2006; Figure 3A). The PHD-finger protein
ING2 tethers the repressive mSin3a-HDAC1 histone de-
acetylases complex to highly active, proliferation-specific
genes after the exposure of cells to DNA-damaging
agents (Pena et al., 2006; Shi et al., 2006). This finding rep-
resents a new mechanism of active shut-off of highly tran-
scribed, H3K4-methylated genes. Two other H3K4me-
binding proteins JMJD2A and CHD1 also tether enzymatic
activities to chromatin, but in these instances the enzy-
matic activity resides within the methyl-binding protein:
JMJD2A is a histone lysine demethylase that binds via
a tudor domain and CHD1 is an ATPase that binds via
a chromodomain (Huang et al., 2006; Pray-Grant et al.,
2005; Sims et al., 2005). One other protein, WDR5, has
been demonstrated to bind H3K4me1 and H3K4me2 (Wy-
socka et al., 2005). However, structural analysis of this in-
teraction does not support a purely methyl-recognition-
based interaction but suggests that this protein binds
most avidly to the residues preceding H3K4 and in partic-
ular to H3R2 (Couture et al., 2006). Perhaps this protein
Figure 2. Crosstalk between Histone
Modifications
The positive influence of one modification over
another is shown by an arrow and the negative
effect by a dish-line.
provides an adaptor function, augmenting the recognition
of H3K4me (Ruthenburg et al., 2006).
Proteins that bind other modified residues also deliver
enzymes: H3K27me recruits the chromodomain contain-
ing polycomb protein PC2, which is associated with
ubiquitin ligase activity specific for H2A; the chromo-
containing HP1 protein binds H3K9me and is associated
with deacetylase activity and methyltransferase activity.
Equally important may be the effectiveness of histone
modifications in preventing the docking of nonhistone
proteins onto chromatin. The study of such pathways is
less detailed, but examples include H3K4me disrupting
the binding of the NuRD complex and H3T3ph preventing
the binding of the INHAT complex. Both complexes
have a repressive capability for transcription, so their
occlusion by positively acting modifications makes sense
(Margueron et al., 2005).
The abundance of modifications on the histone tail
makes ‘‘crosstalk’’ between modifications very likely (Fig-
ure 2). Mechanistically such communication between
modifications may occur at several different levels. Firstly,
many different types of modification occur on lysine resi-
dues (Table 1). This will undoubtedly result in some form
of antagonism since distinct types of modifications on ly-
sines are mutually exclusive. Secondly, the binding of
a protein could be disrupted by an adjacent modification.
The best example of this being that of phosphorylation of
H3S10 affecting the binding of HP1 to methylated H3K9
(Fischle et al., 2005). Thirdly, the catalytic activity of an
enzyme could be compromised by modification of its
substrate recognition site; for example, isomerization of
H3P38 affects methylation of H3K36 by Set2 (Nelson
et al., 2006). Fourthly, an enzyme could recognize its sub-
strate more effectively in the context of a second modifi-
cation; the example here is the GCN5 acetyltransferase,
which may recognize H3 more effectively when it is phos-
phorylated at H3S10 (Clements et al., 2003). Communica-
tion between modifications can also occur when the mod-
ifications are on different histone tails. The best studied
example is the case of ubiquitinilation of H2B being
required for methylation of H3K4me3.
Functional Consequences of Histone Modifications
Simplistically, the function of histone modifications can be
divided into two categories: the establishment of global
chromatin environments and the orchestration of DNA-
based biological tasks. To establish a global chromatin
environment, modifications help partition the genome
into distinct domains such as euchromatin, where DNA
is kept ‘‘accessible’’ for transcription, and heterochroma-
tin, where chromatin is ‘‘inaccessible’’ for transcription. To
facilitate DNA-based functions, modifications orchestrate
the unravelling of chromatin to help the execution of
a given function. This may be a very local function, such
as transcription of a gene or the repair of DNA or it may
be a more genome wide function, such as DNA replication
or chromosome condensation. All these biological tasks
require the ordered recruitment of the machinery to un-
ravel DNA, manipulate it and then put it back to the correct
chromatin state. The term ‘‘histone code’’ has been
loosely used to describe the role of modifications to en-
able DNA functions. This term, although useful in defining
the need for a specific set of modifications for a given task,
is unlikely to truly reflect the presence of a predictable
‘‘code’’ in the strictest sense of the word (Liu et al., 2005).
Below is a brief description of the two categories of func-
tions associated with histone modifications, starting with
the establishment of genomic chromatin environments
followed by the orchestration of processes such as tran-
scription, repair, replication, and chromosome condensa-
tion. (For a detailed discussion of chromatin function
during transcription, DNA replication, and repair, see
Reviews by B. Li et al. and A. Groth et al., pages 707 and
721 of this issue, respectively).
Establishing Global Chromatin Environments
Grossly speaking, there are two different types of chroma-
tin environments in the genome, silent heterochromatin
and active euchromatin. Each of these is associated
with a distinct set of modifications. In mammals, demarca-
tion between the different environments is set up by
boundary elements, which recruit enzymes to modify the
chromatin. The CTCF transcription factor is an example
of a boundary element binding protein that delivers the
modifying enzymes. Experiments in fission yeast have
shown that heterochromatin boundaries are maintained
by the presence of methylation at H3K4 and H3K9 in adja-
cent euchromatic regions. Thus one critical function of
chromatin modifications is that they dictate the different
chromatin environments and preserve these two types
of domains.
Heterochromatin is an important structure, which can
determine the protection of chromosome ends and the
separation of chromosomes in mitosis. In mammals the
Cell 128, 693–705, February 23, 2007 ª2007 Elsevier Inc. 697
silent heterochromatic state is associated with low levels
of acetylation and high levels of certain methylated sites
(H3K9, H3K27, and H4K20). The recruitment of PC2 to
H3K27me is thought to be involved in the maintenance
of the inactive X chromosome, whereas the recruitment
of HP1 to H3K9me is thought to play an important role in
the maintenance of pericentric heterochromatin.
Methylation at H3K27 seems to be missing in both bud-
ding and fission yeast. However H3K9 is present in fission
yeast where heterochromatin is more similar to higher
organisms. In fission yeast there is evidence that the
nucleation of heterochromatin (rather than its spreading)
involves the production of small interfering RNAs (siRNAs)
from transcripts emanating from centromeric repeats. The
dicer-mediated siRNAs are packaged into the RITS com-
plex, which then delivers H3K9 methylation to the sites of
heterochromatin formation. Recruitment of HP1 (Swi6 in
pombe) then allows spreading and maintenance of the
heterochromatic state (Zhang and Reinberg, 2006).
Euchromatin represents a large proportion of the ge-
nome. In this environment DNA has flexibility in biological
output. Genes can be turned on or kept off, DNA can be
‘‘unravelled’’ for repair or replication. Thus the modifica-
tion pattern in euchromatin has to reflect this ‘‘open
choice’’ scenario. In the transcriptionally inactive state,
low levels of acetylation, methylation, and phosphoryla-
tion can be detected on genes, but these are insufficient
to elicit transcription. Further enzymatic activities are nec-
essary for transcription to take place and typically, actively
transcribed euchromatin has high levels of acetylation and
is trimethylated at H3K4, H3K36, and H3K79.
Recently bivalent domains have been found that pos-
sess both activating and repressive modifications, which
somewhat shatters our simplistic view that activating ver-
sus silencing modifications dictate distinct types of chro-
matin environments (Bernstein et al., 2005). Bivalent
domains were discovered during the analysis of numerous
highly conserved noncoding elements in mouse embry-
onic stem cells. The use of ChIP on CHIP technology
revealed that two methylation sites with conflicting output
(H3K27me and H3K4me) coexist in these bivalent do-
mains (Azuara et al., 2006; Bernstein et al., 2005). Classi-
cally H3K27 methylation is implicated in silent chromatin
and H3K4 methylation is involved in active chromatin.
The enrichment of these opposing modifications within
bivalent domains correlated with low-level expression of
developmental transcription factors. However, when ES
cells were made to differentiate, the bivalent domains
tended to preserve either the repressive H3K27me or the
activating H3K4me modification, but not both. The inter-
pretation of these results is that transcription factors that
control certain differentiation processes are kept in
a poised, low-level expression within ES cells by having
a bivalent cluster of modifications. This finding has impor-
tant implications for the preservation of pluripotency in ES
cells. The hope would be that the differentiation of stem
cells can be manipulated by the selective regulation of
modification pathways.
698 Cell 128, 693–705, February 23, 2007 ª2007 Elsevier Inc.
Orchestration of DNA-Based Processes
Transcription
The regulation of gene expression within euchromatin
requires the delivery of chromatin-modifying enzymes by
DNA-bound transcription factors. Following the appear-
ance of a stimulus, transcription factors bind to the
promoter of specific genes and initiate a cascade of mod-
ification events, which result in the expression or silencing
of the gene.
For the purposes of transcription, modifications can be
divided into those that correlate with activation and those
that correlate with repression. Acetylation, methylation,
phosphorylation, and ubiquitination have been implicated
in activation whereas methylation, ubiquitination, sumoy-
lation, deimination, and proline isomerization have been
implicated in repression. However the truth is likely to be
that any given modification has the potential to activate
or repress under different conditions. For example, meth-
ylation at H3K36 has a positive effect when it is found on
the coding region and a negative effect when in the pro-
moter. Methylation at H3K9 may be the same: negative
in the promoter and positive in the coding region (Vakoc
et al., 2005). The more we look in to modifications, the
more it will become clear that context is everything.
In the following few sections, each type of modification
is considered separately regarding its role in transcription,
with emphasis on recently defined functions.
Acetylation. This modification is almost invariably asso-
ciated with activation of transcription. Acetyltransferases
are divided into three main families, GNAT, MYST, and
CBP/p300 (Sterner and Berger, 2000). In general these
enzymes modify more than one lysine but some limited
specificity can be detected for some enzymes (Table 2).
Most of the acetylation sites characterized to date fall
within the N-terminal tail of the histones, which are more
accessible for modification. However, a lysine within the
core domain of H3 (K56) has recently been found to be
acetylated. A yeast protein SPT10 may be mediating acet-
ylation of H3K56 at the promoters of histone genes to
regulate gene expression (Xu et al., 2005), whereas the
Rtt109 acetyltransferase mediates this modification
more globally (Han et al., 2007; Driscoll et al., 2007;
Schneider et al., 2006). The K56 residue is facing toward
the major groove of the DNA within the nucleosome, so
it is in a particularly good position to affect histone/DNA
interactions when acetylated.
Deacetylation. The reversal of acetylation correlates
with transcriptional repression. There are three distinct
families of histone deacetylases: the class I and class II
histone deacetylases and the class III NAD-dependant
enzymes of the Sir family. They are involved in multiple
signaling pathways and they are present in numerous
repressive chromatin complexes. In general these en-
zymes do not appear to show much specificity for a partic-
ular acetyl group although some of the yeast enzymes
have specificity for a particular histone: Hda1 for H3 and
H2B; Hos2 for H3 and H4. The fission yeast class III de-
acetylase Sir2 has some selectivity for H4K16ac, and
recently the human Sir family member SirT2 has been
demonstrated to have a similar preference (Vaquero
et al., 2006).
Phosphorylation. Little is known about histone phos-
phorylation and gene expression. MSK1/2 and RSK2 in
mammals, and SNF1in budding yeast, have been shown
to target H3S10. A role for H3S10 phosphorylation has
been demonstrated for the activation of NFKB-regulated
genes and also ‘‘immediate early’’ genes such as c-fos
and c-jun. Concomitant with this phosphorylation is the
appearance on chromatin of a phosphor-binding protein
14-3-3 (Macdonald et al., 2005). Recently, a global ChIP
on CHIP analysis of many kinases in budding yeast has
shown that they are present on the chromatin of specific
genes (Pokholok et al., 2006). This has important implica-
tions regarding signal transduction. It suggests that the
mainly cytoplasmic protein phosphorylation cascades
that have dominated signal transduction processes for
many years may have a more direct effect on gene expres-
sion through the phosphorylation of chromatin.
Lysine Methylation. Lysine methyltransferases have
enormous specificity compared to acetyltransferases
(Table 2). They usually modify one single lysine on a single
histone and their output can be either activation or repres-
sion of transcription (Bannister and Kouzarides, 2005).
Three methylation sites on histones are implicated in ac-
tivation of transcription: H3K4, H3K36, and H3K79. Two of
these, H3K4me and H3K36me, have been implicated in
transcriptional elongation. In budding yeast H3K4me3
localizes to the 50 end of active genes and is found asso-
ciated with the initiated form of RNA Pol II (phosphorylated
at serine 5 of its C-terminal domain). H3K36me3 is found
to accumulate at the 30end of active genes and is found
associated with the serine 2 phosphorylated elongating
form of RNA pol II. One role for H3K36me is the suppres-
sion of inappropriate initiation from cryptic start sites
within the coding region (Carrozza et al., 2005; Cuthbert
et al., 2004; Joshi and Struhl, 2005; Keogh et al., 2005).
To achieve this, methylation at H3K36 recruits the EAF3
protein, which in turn brings the Rpd35 deacetylase com-
plex to the coding region. Deacetylation then removes any
acetylation that was placed in the coding region during the
process of transcription, thus resetting chromatin into its
stable state. This ‘‘closing up’’ of chromatin, following
the passage of RNA pol II, prevents access of internal ini-
tiation sites that may be inappropriately used. Very little is
known about the function of methylation at H3K79. We do
know that it is involved in the activation of HOXA9 and
it has a role in maintaining heterochromatin, probably
indirectly, by limiting the spreading of the Sir2 and Sir3
proteins into euchromatin.
Three lysine methylation sites are connected to tran-
scriptional repression: H3K9, H3K27, and H4K20. Methyl-
ation at H3K9 is implicated in the silencing of enchromatic
genes as well as forming silent heterochromatin men-
tioned above. Repression involves the recruitment of
methylating enzymes and HP1 to the promoter of
repressed genes. Delivery of these components of meth-
ylation-based silencing is mediated by corepressors such
as RB and KAP1. The dogma, that H3K9 methylation and
HP1 recruitment are always repressive, has recently been
challenged by the finding that H3K9me3 and the g isoform
of HP1 are enriched in the coding region of active genes
(Vakoc et al., 2005). The explanation for this difference is
not clear. One possibility is that H3K9me within the coding
regions is activatory whereas H3K9me in the promoters
is repressive.
H3K27 methylation has been implicated in the silencing
of HOX gene expression. A similar mechanism is likely to
be operational for the involvement of H3K27me in silenc-
ing of the inactive X chromosome and during genomic im-
printing. Very little is known regarding the repression func-
tions of H4K20 methylation. It has a role in the formation of
heterochromatin and has a role in DNA repair. Recently
a protein has been identified that may mediate its func-
tions. The JMJD2A lysine demethylase has been demon-
strated to bind H3K20me (Huang et al., 2006; Kim et al.,
2006) via a tudor domain. The implications of this interac-
tion are not clear especially given that JMJD2A can also
bind the positively acting methylation site at H3K4.
Lysine Demethylation. For a number of years following
the discovery of histone methyltransferases, the existence
of demethylases was contentious. The discovery of the
first histone demethylase LSD1 (Shi et al., 2004) has
opened the way for the discovery of many other such en-
zymes (Table 2). So far there are two types of demethylase
domain, with distinct catalytic reactions: the LSD1 domain
and the JmjC domain. LSD1 acts to demethylate H3K4
and repress transcription (Shi et al., 2004). However
when LSD1 is present in a complex with the androgen re-
ceptor, it demethylates H3K9 and activates transcription
(Metzger et al., 2005). H3K9 can also be demethylated
by JHDM2A (Yamane et al., 2006), JMJD2A/JHDM3A
(Tsukada et al., 2006; Whetstine et al., 2006), JMJD2B
(Fodor et al., 2006), JMJD2C/GASC1 (Cloos et al., 2006),
and JMJD2D (Shin and Janknecht, 2006). Methylation at
H3K36 can be reversed by JHDM1 (Tsukada et al.,
2006; Whetstine et al., 2006), JMJD2A/JHDM3A (Klose
et al., 2006), and JMJD2C/GASC1 (Cloos et al., 2006).
Structural analysis of JMJD2A has shown that three dis-
tinct domains, in addition to the JmjC domain, are neces-
sary for catalytic activity (Chen et al., 2006).
It is too early to know the precise function of all these new
demethylases. What is clear is that they will antagonize
methylation by being delivered to the right place at the right
time (Yamane et al., 2006). Also, the activity of the enzymes
are under the influence of the proteins they bind, as in the
case of LSD1/BHC110, which acts on nucleosomal sub-
strates in the presence of CoREST (Lee et al. 2005a). A
very important part of the specificity of these new deme-
thylases also comes down to the state of methylation
they act on. Their selectivity for mono-, di-, or trimehylated
lysines allows for a larger functional control of lysine meth-
ylation (Shi and Whetstine, 2007).
Arginine Methylation. Like lysine methylation, arginine
methylation can be either activatory or repressive for
Cell 128, 693–705, February 23, 2007 ª2007 Elsevier Inc. 699
transcription, and the enzymes (protein arginine methyl-
transferases, PRMT’s) are recruited to promoters by
transcription factors (Lee et al., 2005b). The most studied
promoter regarding arginine methylation is the estrogen-
regulated pS2 promoter. A very interesting observation
regarding this promoter is that modifications are cycling
(appear and disappear) during the activation process
(Metivier et al., 2003). The reason for this is not known,
and certainly this is not a behavior represented at most
other genes. The reason may be that estrogen-regulated
genes have to respond to outside stimuli very rapidly, so
their chromatin has to be in ‘‘a state of alert’’ for impending
shutdown of transcription. There are no proteins yet
identified that can bind specifically to arginine-methylated
histones and no enzymes that can reverse arginine
methylation.
Deimination. This involves the conversion of an arginine
to a citrulline. Arginines in H3 and H4 can be converted to
citrullines by the PADI4 enzyme. Deimination has the
potential to antagonize the activatory effect of arginine
methylation since citrulline prevents arginines from being
methylated (Cuthbert et al., 2004; Wang et al., 2004a). In
addition, in vivo data demonstrate that mono- (but not
di-) methylated arginines can be deiminated (Wang
et al., 2004a). In vitro analysis of the PADI4 enzyme sug-
gests that the reversal of monomethyl arginine to citrulline
is not carried out by the recombinant enzyme when meth-
ylated peptides are used as substrates, suggesting that
a cofactor may be necessary in vivo (Hidaka et al.,
2005). Converting citrulline to arginine has not been
described, although citrulline is cyclic on the pS2
promoter, so reversal may be possible (Bannister and
Kouzarides, 2005).
Ubiquitylation. This very large modification has been
found on H2A (K119) and H2B (K20 in human and K123
in yeast). Ubiquitylation of H2AK119 is mediated by the
Bmi/Ring1A protein found in the human polycomb com-
plex and is associated with transcriptional repression
(Wang et al., 2006). This modification is not conserved in
yeast. In contrast, H2BK120 ubiquitylation is mediated
by human RNF20/RNF40 and UbcH6 and in budding
yeast by Rad6/Bre1 and is activatory for transcription
(Zhu et al., 2005). A role for this modification has been
demonstrated in transcriptional elongation by the histone
chaperone FACT (Pavri et al., 2006). How ubiquitylation
functions is unclear; it is likely to recruit additional factors
to chromatin but may also function to physically keep
chromatin open by a ‘‘wedging’’ process, given its large
size.
Deubiquitylation. In budding yeast, two enzymes (Ubp8
and Ubp10) have been identified that antagonize ubiquity-
lation of H2BK123. The Ubp8 enzyme (subunit of the
SAGA acetyltransferase complex) is required for activa-
tion of transcription, indicating that both the addition and
removal of ubiquition is necessary for stimulation of tran-
scription. The Ubp10 deubiquitylase functions in tran-
scriptional silencing at heterochromatic sites in budding
yeast (Emre et al., 2005; Gardner et al., 2005).
700 Cell 128, 693–705, February 23, 2007 ª2007 Elsevier Inc.
Sumoylation. Like ubiquitylation, sumoylation is a very
large modification and shows some low similarity to ubiq-
uitylation. This modification has been shown to take place
on all four core histones, and specific sites have been
identified on H4, H2A, and H2B (Nathan et al., 2006, #2).
Sumoylation antagonizes both acetylation and ubiquityla-
tion, which occur on the same lysine residue, and conse-
quently this modification is a repressive one for transcrip-
tion in yeast.
ADP Ribosylation. This histone modification is ill defined
with respect to function. ADP ribosylation can be mono- or
poly-, and the enzymes that mediate it are MARTs (Mono-
ADP-ribosyltransferases) or PARPs (poly-ADP-ribose
polymerases), respectively (Hassa et al., 2006). In addition
the Sir family of NAD-dependent histone deacetylases
have been shown to have low levels of this activity, so
they may represent another class of this family. There are
many reports of ADP ribosylation of histones, but only
one site, H2BE2ar1, has been definitively mapped. Al-
though the function of the enzymes has often been linked
to transcription, evidence that the catalytic activity is in-
volved has been lacking. Recently a role for PARP-1 activ-
ity in transcription has been demonstrated but only under
conditions where DNA repair is induced. Double-strand
breaks mediated by Topoisomerase II b activate the
PARP-1 enzyme, which then directs chromatin changes
to the estrogen-regulated PS2 gene (Ju et al., 2006).
Proline Isomerization. Prolines exist in either a cis or
trans conformation. These conformational changes can
severely distort the polypeptide backbone. Recently an
enzyme, FPR4, has been identified in budding yeast that
can isomerize prolines in the tail of H3 (Nelson et al.,
2006). FPR4 isomerizes H3P38 and thereby regulates
the levels of methylation at H3K36. The appropriate pro-
line isomer is likely to be necessary for the recognition
and methylation of H3K36 by the Set2 methyltranferase.
In addition, it is possible that demethylation of H3K36 is
also affected by isomerization at H3P38 (Chen et al.,
2006). The catalytic cleft of the JMJD2 demethylase is
very deep and may necessitate a bend in the polypeptide
(mediated by proline isomerization) to accommodate the
methyl group at H3K36.
DNA Repair
Phosphorylation. Chromatin generates a barrier for the
repair of DNA damage. Modifications on histones assist
in the recognition and accessibility of sites where DNA
repair needs to take place. One of the earliest recognized
responses to DNA damage is the phosphorylation of the
histone variant g-H2AX in mammalian cells (Fillingham
et al., 2006). This phosphorylation extends over many kilo-
bases around the site of the damage. In budding yeast
phosphorylation of H2AX has been shown to recruit
the INO80 complex, which possesses ATP-dependant re-
modeling activity (Van Attikum et al., 2004). Two phos-
phorylation sites on this histone have a role in double-
strand break repair via nonhomologous end joining:
H2AS129 mediated by Mec1 (Downs et al., 2000) and
H4S1 mediated by Caesin kinase II (Cheung et al., 2005).
Methylation. In fission yeast, ionizing radiation-induced
DNA damage generates nuclear foci at sites of DNA repair,
which contain methylated H4K20 and the cell-cycle
checkpoint protein Crb2 (Sanders et al., 2004). This pro-
tein signals a G2/M arrest in order for the DNA to be re-
paired (Figure 3B). Crb2 recruitment to DNA repair foci is
dependant on the recognition of methylated H4K20 via
the double tudor domains of Crb2 (Botuyan et al., 2006).
Methylation at H4K20 is present throughout the genome.
During DNA damage it becomes ‘‘apparent’’ at the sites
of DNA repair but appears absent elsewhere. So how
does Crb2 recruitment take place so specifically at these
sites? The answer may lie in a second modification,
a phosphorylation of H2AX that Crb2 recognizes at these
sites via its BRCT domain. This phosphor-binding domain
may recognize the DNA-damage-induced phosphoryla-
tion site and then stabilize itself on chromatin via the rec-
ognition of H4K20me (Du et al., 2006). In human cells,
Figure 3. Functional Consequences of Histone Modifications
(A) Gene-expression changes are brought about by the recruitment of
the NURF complex, which contains a component BRTF recognizing
H3K4me and a component-remodeling chromatin.
(B) The Crb2 protein of fission yeast is recruited to DNA-repair foci dur-
ing a DNA-repair response. Crb2 is partly tethered there by association
with methylated H4 and phosphorylated H2A.
(C) The HBO1 acetyltransferase is an ING5-associated factor and is
therefore tethered to sites of replication via methylated H3K4. HBO1
also binds to the MCM proteins found at replication sites. Evidence ex-
ists that HBO1 augments the formation of the preinitiation complex
and is required for DNA replication.
p53BP1, the homolog of Crb2, may operate in a very sim-
ilar way. Although this protein may have some affinity for
H3K79 methylation (Huyen et al., 2004), recent structural
and functional studies suggest that this protein recognizes
H4K20 methylation very avidly and is recruited to sites of
DNA via H4K20 methylation (Botuyan et al., 2006). Inter-
estingly, Crb2 and p53BP1 only recognize the mono-
and dimethyl forms of H4K20, which opens the possibility
that the trimethyl form may function to regulate a different
step in DNA repair, or it may be involved in a completely
different function in the absence of DNA-damage signal-
ing.
Acetylation. In budding yeast acetylation of H3K56 is
deposited on newly synthesized histones during S phase.
In the absence of damage, H3K56 acetylation disappears
in G2. However, in the presence of DNA damage the de-
acetylases for H3K56, Hst3, and Hst4 (two paralogs of
Sir2) are downregulated and the modification persists
(Celic et al., 2006; Maas et al., 2006). The Rtt109 enzyme,
which acetylates H3K56, has recently been implicated in
genome stability and DNA replication (Driscoll et al.,
2007; Han et al., 2007; Schneider et al., 2006). The yeast
acetyltransferase Hat1 is another enzyme that is impli-
cated in DNA repair. This enzyme is recruited to sites of
DNA repair and acetylates H4K12 (Qin and Parthun, 2006).
Ubiquitination. This is the most recent modification to
be linked to DNA repair. UV-induced DNA repair signals
ubiquitination of H3 and H4 by the CUL4-DDB-Roc1 com-
plex (Wang et al., 2006). Misregulation of this ubiquition
ligase complex by downregulation of CUL4A prevents
the recruitment of the XPC repair protein to DNA-damage
foci. Monoubiquitylation of H2A is also implicated in
UV-induced repair (Bergink et al., 2006). In this case, the
Ring2 ubiquition ligase mediates the modification. The
monoubiquitylation of H2A is coincident with H2AX phos-
phorylation but is independent of it. Instead, a DNA-
damage-specific kinase, ATM, seems to be necessary
for this modification to take place.
DNA Replication
Acetylation. A role for acetylation in DNA replication was
suspected some time ago when an acetyltransferase,
HB01, was isolated as a binding partner for an origin
recognition complex protein. More recently a very central
role for HB01 in DNA replication has emerged. In the pro-
cess of analyzing the stoichiometric partners of the ING
family of proteins, HB01 was found in a complex with
ING4 (a tumor suppressor) and ING5 (Doyon et al.,
2006). Depletion of ING5 and depletion of HB01, although
less severe, causes a reduction of DNA synthesis and
affects progression into S phase. In a separate study
HB01 is shown to augment the assembly of the pre-repli-
cative complex and the recruitment of MCMs to chromatin
(Iizuka et al., 2006). In Drosophila, the HB01 homolog,
Chameau, is found to increase the firing of replication
origins (Aggarwal and Calvi, 2004). Together, these find-
ings suggest that HBO1, via its ability to acetylate H4, is
required for S phase initiation and fixing of replication
origins (Figure 3C).
Cell 128, 693–705, February 23, 2007 ª2007 Elsevier Inc. 701
Chromosome Condensation
Phosphorylation. Condensation and decondensation of
chromatin are important processes during the replicative
cell cycle. Two phosphorylation events in mammalian
cells may play an important role in these processes during
mitosis. The first is phosphorylation of H3S10 during mito-
sis by the Aurora B kinase. Recent data suggest that one
of the mechanisms by which H3S10 phosphorylation may
function is via the displacement of HP1 from H3K9me,
which normally compacts chromatin (Fischle et al.,
2005). The second phosphorylation event is at H3T3
(Dai et al., 2005). This modification is mediated by the Has-
pin kinase and is required for normal metaphase chromo-
some alignment. A number of other phosphorylation sites
have been implicated in this process in budding yeast.
Phosphorylation of H4S1 regulates sporulation (Krishna-
moorthy et al., 2006), and phosphorylation of H2BS10 reg-
ulates peroxide-induced apoptosis (Ahn et al., 2005). The
latter modification is on a residue that is not conserved
in mammals. However, phoshorylation of mammalian
H2BS14 by Mst1 is thought to play an analogous function.
Acetylation. In vitro experiments provide a role for
H4K16Ac in chromatin decondensation (Shogren-Knaak
et al., 2006). A class III deacetylase SirT2, which has spec-
ificity for H4K16Ac, may have the ability to induce the
condensation of chromatin in vivo (Vaquero et al., 2006).
Consistent with this idea is the finding that SirT2 localizes
to chromatin during G2/M transition when chromatin has
to be recondensed.
Are Histone Modifications Truly Epigenetic?
Histone modifications have been implicated in a number
of epigenetic phenomena. The classic definition of epige-
netics is the study of heritable phenotype changes that do
not involve alterations in DNA sequence. The use of the
term ‘‘heritable’’ has been dropped in recent usage, allow-
ing the term epigenetic to mean the information carried by
the genome (e.g., on chromatin) that is not coded by DNA.
However the classic term, that includes heritability, is im-
portant to maintain as it defines a nongenetic memory of
function that is transmitted from generation to generation.
A number of cellular phenotypes are transmitted in this
way, including imprinting, X chromosome inactivation, ag-
ing, heterochromatin formation, reprogramming, and
gene silencing. In addition there are environmentally in-
duced changes, which are passed on from generation to
generation, without the need for the original stimulus
(most studied in plants). There is no disputing that histone
modifications are involved in epigenetic processes. The
question is, do modifications pass on the memory of
a given chromatin state or do they merely implement the
memory, once the memory is passed on via a distinct
process?
If epigenetic memory is mediated by one or more of the
histone modifications, then there should be a mechanism
for the transmission of such modifications onto the chro-
matin of the replicating DNA. Such a mechanism has
been proposed for H3K9 methylation in the transmission
702 Cell 128, 693–705, February 23, 2007 ª2007 Elsevier Inc.
of the heterochromatin: recruitment of HP1 brings in fur-
ther H3K9-methylating activity that modifies nucleosomes
on the daughter strand, thus ensuring the transmission of
the H3K9me mark. This mechanism of transmission, along
with the observation that H3K4me3 patterns persist, have
given lysine methylation an epigenetic status. The issue
that remains, however, is whether the modification pattern
inherited by the daughter chromatin is sufficient to impose
the correct chromatin structure originating from the
mother cell. Is methylation of lysines dictating the memory
of chromatin structure?
The argument that histone methylation is a permanent
mark is now on shaky ground, given the discovery of de-
methylases. Are other types of histone modifications epi-
genetic? Do we expect the complicated chromatin struc-
ture of the entire genome to be perpetuated by a few
inherited histone modifications? Are there other determi-
nants likely to transmit information for the assembly of
a correct local chromatin structure?
One such determinant is RNA. Work in fission yeast has
shown that small RNAs are associated with chromatin-
modifying complexes and can deliver histone-modifying
enzymes to chromatin (Verdel et al., 2004). Deletion of
the enzyme Dicer that prosesses small RNAs can also af-
fect heterochromatin formation, methylation of H3K9, and
recruitment of HP1 (Fukagawa, 2004; Kanellopoulou et al.,
2005; also see Review by M. Zaratiegui et al., page 763
of this issue).
The case for RNA as a determinant is certainly appeal-
ing, and some evidence exists that it acts in such a way.
Recent work in mice has shown that small RNAs present
in sperm can be transmitted to offspring where they medi-
ate an epigenetic phenotype called paramutation, a
process first identified in plants (Rassoulzadegan et al.,
2006). Perhaps this mechanism is more widespread
than we think. Small RNAs may emanate from many
loci in the genome and once transmitted to the next gen-
eration, these RNAs may deliver chromatin-modifying
complexes to specific genes or to specific locations,
thus generating the pattern of chromatin that we observe
(Verdel et al., 2004; Buhler et al., 2006). One appealing
aspect of this model is that small RNAs are likely to be
highly precise in their delivery since their guiding system
is nucleic acid.
Only time will tell whether such speculative mechanisms
exist for the widespread transfer of chromatin information.
The model proposed implies that RNA may be perfect as
a molecule to transmit the memory of a specific chromatin
state. However, such an RNA-mediated mechanism does
not imply that histone modifications are unnecessary for
epigenetic events. It merely points out that histone modi-
fications may be the executers of the epigenetic phenom-
enon rather than the carriers of the memory.
ACKNOWLEDGMENTS
I thank Andy Bannister for helpful discussions. T.K. is a director of
Abcam plc.
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